Rapid screening of point mutations by mismatch amplification mutation assay PCR

Abstract Metabolic engineering frequently makes use of point mutation and saturation mutation library creation. At present, sequencing is the only reliable and direct technique to detect point mutation and screen saturation mutation library. In this study, mismatch amplification mutation assay (MAMA) PCR was used to detect point mutation and screen saturation mutation library. In order to fine-tune the expression of odhA encoding 2-oxoglutarate dehydrogenase E1 component, a saturating mutant library of the RBS of odhA was created in Corynebacterium glutamicum P12 based on the CRISPR-Cas2a genome editing system, which increased the l-proline production by 81.3%. MAMA PCR was used to filter out 42% of the non-mutant transformants in the mutant library, which effectively reduced the workload of the subsequent fermentation test and the number of sequenced samples. The rapid and sensitive MAMA-PCR method established in this study provides a general strategy for detecting point mutations and improving the efficiency of mutation library screening. Key points • MAMA PCR was optimized and developed to detect point mutation. • MAMA PCR greatly improves the screening efficiency of point mutation. • Attenuation of odhA expression in P12 effectively improves proline production. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-024-13036-2.


Construction of plasmids and plasmid library.
For the construction of plasmid pFST-proB-proB G149K , the fragment obtained by annealing primer sgRNA-proB-F/R was digested by EcoR I/Hind III and cloned into pFST to obtain plasmid pFST-proB.
The primers proB G149K -F1/R1 and proB G149K -F2/R2 were amplified by C. glutamicum ATCC 13032 genome to obtain fragments proB G149K -1 and proB G149K -2, respectively.The primer pFST-vector-F/R was amplified with pFST-proB as the template to obtain the fragment pFST-proB-vector; Finally, the three fragments were ligated by the ClonExpress MultiS One Step Cloning Kit to obtain plasmid pFST-proB-proB G149K .
For the construction of plasmid pFST-gRNA1, the fragment obtained by annealing primer sgRNAzwf-1-F/ sgRNA-proB-R was digested by EcoR I/Hind III and cloned into pFST to obtain plasmid pFST-gRNA1.
For the construction of plasmid pFST-gRNA2, the fragment obtained by annealing primer sgRNAzwf-2-F/ sgRNA-proB-R was digested by EcoR I/Hind III and cloned into pFST to obtain plasmid pFST-gRNA2.
For the construction of plasmid pFST-gRNA4, the fragment obtained by annealing primer sgRNAgnd-F/ sgRNA-proB-R was digested by EcoR I/Hind III and cloned into pFST to obtain plasmid pFST-gRNA4.
For the construction of plasmid library pJYS3_0, the primer pJYS3_0-F/R was used to whole plasmid PCR using plasmid pJYS3_crtYf as template, and the PCR product was digested by Dpn I, and then purified.Then, the purified fragment was ligated by the ClonExpress II One Step Cloning Kit to obtain plasmid pJYS3_0.
For the construction of plasmid library pJYS3_gRNA3, the primer crRNA-zwf-F/R was used to whole plasmid PCR using plasmid pJYS3_crtYf as template, and the PCR product was digested by Dpn I, and then purified.Then, the purified fragment was ligated by the ClonExpress II One Step Cloning Kit to obtain plasmid pJYS3_gRNA3.
For the construction of plasmid library pJYS3_gRNA5, the primer crRNA-gnd-F/R was used to whole plasmid PCR using plasmid pJYS3_crtYf as template, and the PCR product was digested by Dpn I, and then purified.Then, the purified fragment was ligated by the ClonExpress II One Step Cloning Kit to obtain plasmid pJYS3_gRNA5.
For the construction of plasmid library pJYS3_odhARBS-LIB, the primer crRNA-odhARBS-F/R was used to whole plasmid PCR using plasmid pJYS3_crtYf as template, and the PCR product was digested by Dpn I, and then purified.Then, the purified fragment was ligated by the ClonExpress II One Step Cloning Kit to obtain plasmid pJYS3_odhA RBS .The primer odhA RBS -F/R was amplified by P12 genome to obtain the fragment odhA RBS ; The primer pJYS3-vector-F/R was amplified by using pJYS3-odhA RBS as a template to obtain a fragment pJYS3-odhA RBS -vector; Subsequently, the two fragments were ligated by the ClonExpress II One Step Cloning Kit to obtain plasmid pJYS3_odhA RBS -HD odhA .
Then, the whole plasmid PCR was carried out by primer odhA RBS -LIB-F/R with plasmid pJYS3_odhA RBS -HD odhA as template, and the PCR product was digested by Dpn I, and then purified.
Finally, the purified fragment was ligated by the ClonExpress II One Step Cloning Kit and transformed into E. coli JM109 competent, and the plasmid library pJYS3_odhA RBS -LIB was obtained.
For the construction of plasmid library pK18-Zwf A243T , the upstream and downstream gene fragments of zwf were amplified from genomic DNA by primers Zwf A243T -up-F/R and Zwf A243T -down-F/R respectively, and then the two fragments were fused by overlapping extension PCR to obtain DNA fragment Zwf A243T -UD.Subsequently, Zwf A243T -UD was inserted into Hind III/Xba I-digested pK18mobsacB to obtain pK18-Zwf A243T .
For the construction of plasmid library pK18-Gnd S361F , the upstream and downstream gene fragments of gnd were amplified from genomic DNA by primers Gnd S361F -up-F/R and Gnd S361F -down-F/R respectively, and then the two fragments were fused by overlapping extension PCR to obtain DNA fragment Gnd S361F -UD.Subsequently, Zwf A243T -UD was inserted into Xba I/BamH I-digested pK18mobsacB to obtain pK18-Zwf A243T .DNA sequencing was used to verify the plasmids.Table S1.Sequencing results of proB site-directed mutation transformants edited by CRISPR-Cas9 system

Figure S1 .
Figure S1.Detection of point mutation ProB G149K by MAMA PCR. (a) Sequencing results of proB

Figure S2 .
Figure S2.Detection of point mutation ilvC TM by MAMA PCR.△LtbR and △LtbRAHAIR M are

Figure S3 .
Figure S3.Activity determination of gRNA (guide RNA) at the mutation site of zwf and gnd.(a)

Figure S4 .
Figure S4.Effect of L-glutamate addition on L-proline fermentation in P12 strain.Error bars

Gnd
The bold letters in red represent the mismatched bases on the MAMA PCR primer with the unmutated template.

Table S3 .
L-proline fermentation of transformants screened by MAMA PCR from the RBS